SUMMARY:The sonic disruption of spores of Bacillus cereus gives multi-hit kinetics. The flrst hit destroys the exosporium, which protects the spore body from destruction ; the second hit destroys the spore body. Spores which have been stripped oftheir exosporia are still viable and heat resistant. From the rate of release during genic tmatment it appears that alanine raceITLsse, adenosine deaminase and hexosamine are located in the exosporium while ribosidase is in the spore body. The rate of release of dipicolinic acid does not identify it$ location.Since the discovery of alanine racemase in spores (Stewart & Halvorson, 1953) several other enzymes have been found in preparations of well-cleaned spores (reviewed by Halvorson & Church, 1957). Many of the enzymes in extracts of spores are particulate and heat-stable, but the location of the enzymes within the spore is somewhat obscure. Electron micrographs of thin sections of spores of BaciZZzM cmew (Robinow, 1958) show a large well-defined exosporium as the outermost structure surrounding the spore coat. The electrondense spore coat, in turn, delimits what we shall refer to as the spore body. The particulate enzymes of the spore could be contained within the spore body or could be submicroscopic fragments of the spore coat or of the exosporium. A decision between similar alternatives in Axotobmter virtelartdii has been made by measuring the rates of release of constituents during sonic disruption (Marr & Cota-Robles, 1957). Submicroscopic particles and soluble constituents of the cytoplasm were released at the same rate as the disruption of the cell, while submicroscopic particles derived by disintegration of the envelope were released more slowly. In the application of this technique of Merential release to the biochemical cytology of spores we were somewhat surprised to find that several constituents of the spore are released more Tap'dg than the rate of disruption of the spore body. These constituents appear to be in the exosporium. METHODSOrganism and w h r a l metho&. B Spores were harvested in a Sharpies centrifuge and washed 6-10 times with distilled water or phosphate buffer until microscopic examination indicated the absence of debris from vegetative cells. The spores were suspended in 0.05 M-phosphate buffer (pH 6.8) to a concentration of 8 x loo spores/ml. with less than 5 % gemhated spores. Sm& disruptim. Of the above spore suspension 50ml. were treated at 75 scousticd watb in a Raytheon 10 kc. sonic oscillator at 0 ' 4 ' and in a gas atmosphere of H , . At various times, 8 or 5 ml. samples were removed and were replaced by an equal volume of buffer. Corrections were made for the progressive dilution resulting from this sampling. Release of constituents was determined by centrifuging portions of the samples at 10,WO.g for 15 min., which was sufficient to sediment residual spores and fragments of microscopic dimensions. Release was judged by failure to sediment in this centrifugation. All assays except that for deaminase were made on the residue.Tu...

Countermeasures to biofouling in simulated ocean thermal energy conversion heat exchangers have been studied in single-pass flow systemis, hsing cold deep and warm surface ocean waters off the island of Hawaii. Manual brushing of the loops after free fouling periods removed most of the biofouling material. However, over a 2-year period a tenacious film formed. Daily free passage of sponge rubber balls through the tubing only removed the loose surface biofouling layer and was inadequate as a countermeasure in both titanium and aluminum alloy tubes. Chlorination at 0.05, 0.07, and 0.10 mg liter-' for 1 h day'1 lowered biofouling rates. Only at 0.10 mg liter-' was chlorine adequate over a 1-year period to keep film formation and heat transfer resistance from rising above the maximum tolerated values. Lower chlotination regimens led to the buildup of uneven or patchy ifims which produced increased flow turbulence. The result was loWver heat transfer resistance values which did not correlate with the amount of biofouling. Strfaces which were let foill and then treated With intermittent or continuous chlorination at 0.10 mg of chlorine or less per liter were only partially of unevenly cleaned, although heat transfer measurements did not indicate that fact. It took cdntihubus chlorination at 0.25 mg liter-' to bring the heat transfer resistance to zero and eliminate the fouling layer. Biofouling in deep coid seawater was much slower than in the warm surface waters. Tubing in one stainless-steel loop had a barely detectable fouling layer after 1 year in flow. With aluminum alloys sufficient corrosion and biofouling material accumulated to require that some fouling coutermeasure be used in long-term operation of an ocean thermal energy conversion plant.

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